5,10-methylene-tetrahydrofolate as a modulator of a chemotherapeutic agent

ABSTRACT

The present invention relates to the compound 5,10-methylene-tetrahydrofolate (CH 2  FH 4 ), and its solution product isomer FH 4 , therapeutic uses of these compounds, and compositions thereof. CH 2  FH 4  and FH 4  strongly modulate the in vivo antitumor effects of 5-Fluorouracil.

This is a continuation of Ser. No. 07/789,729 filed Nov. 12, 1991, nowabandoned, which in turn is a continuation-in-part of Ser. No.07/521,712 filed May 11, 1990, now abandoned.

BACKGROUND OF THE INVENTION

1. Technical Field

The subject matter of the present invention relates to5,10-methylene-tetrahydrofolate (CH₂ FH₄), therapeutic uses of thiscompound and compositions thereof. CH₂ FH₄ strongly modulates the invivo antitumor effects of 5-Fluorouracil. Furthermore, the presentinvention additionally relates to a solution product isomer of CH₂ FH₄,tetrahydrofolate (FH₄), which also strongly modulates the in vivoantitumor effects of 5-Fluorouracil. CH₂ FH₄ and FH₄ both exist in anatural ((6R)-CH₂ FH₄ and (6S)-FH₄) and unnatural ((6S)-CH₂ FH₄ and(6R)-FH₄) diastereomeric form and both forms may be utilized forpurposes of the present invention. In solution, CH₂ FH₄ and FH₄ exist inchemical equilibrium, with requirements for millimolar formaldehydeconcentrations to favor the balance toward CH₂ FH₄.

2. Background Information

The compound 5-Fluorouracil (5-FU) is possibly the most widely usedanticancer drug in the world. In the 1970s and early 1980s, theprevailing opinion among cancer researchers was that the key biochemicallesion caused by 5-FU in tumor cells resulted from the drug'sincorporation into RNA (Kufe et al., J. Biol. Chem. 256:9802 (1981) andGlazer et al., Mol. Pharmacol. 21:468 (1982)).

In 1982, using a specifically designed assay of the DNA enzyme,thymidylate synthase (TS) (EC 2.1.1.45), the present inventorsestablished that the therapeutic mechanism of 5-FU against murine coloncancer was complete inhibition of TS or abrogation of TS activity(Spears et al., Cancer Res. 42:450-56 (1982)). In fact, the presentinventors were the first to report a clinical correlation between TSlevel in a patient's cancer after 5-FU treatment and response (Spears etal., Cancer Res. 44:4144-50 (1984)). The finding has been confirmed byseveral research groups.

TS is the only intracellular source of new ("de novo") thyminesynthesis, as the enzyme which catalyzes the methylation ofdeoxyuridylate to form thymidylate(thymine-2'-deoxyribose-5'-phosphate). Thymine is one of the four mainbuilding blocks of DNA, and its occurrence in DNA (vs. its absence inRNA) is the major structural difference between DNA and RNA. Thus, theactivity of TS to make new thymidylate and DNA is essential to celldivision, tissue regeneration and turnover, and tumor growth. The sourceof the methyl one-carbon group for synthesis of thymidylate is CH₂ FH₄and its polyglutamates. The mechanism of methyl transfer by TS hasrecently been reviewed (K. T. Douglas, Medicinal Res. Rev. 7:441-75(1987)). After initial weak binding of deoxyuridylate to TS, the enzymecatalyzes ring-opening of CH₂ FH₄ at the imidazole C11 ring. This may bethe rate limiting step overall. The relative stability oftetrahydrofolate within the ternary complex, toward oxidation, suggeststhat the ring-opening occurs with the substitution at N5, in accordancewith formation of an N5-iminium cation species (S. J. Benkovic, Ann.Rev. Biochem., 49:227- 51 (1980)). Covalent bonding between themethylene group and the C5-position of deoxyuridylate is accompanied byrapid hydride transfer from the C6-position of the ring-opened CH₂ FH₄so that CH3- is formed on the C5 position of the nucleotide. This leadsrapidly to expulsion of the two products from the TS binding site(s),i.e., thymidylate and dihydrofolate. TS is the only enzyme whichoxidizes reduced folates to dihydrofolate, which is then converted backto tetrahydrofolate by another enzyme, dihydrofolate reductase. Ingeneral, the limiting intracellular factors in this biochemical pathwayfor making thymine are, in order of increasing scarcity, deoxyuridylate,dihydrofolate reductase, TS, and then CH₂ FH₄. Thus, a decrease inthymidine production through the TS pathway can result from nutritionaldeficiencies which decrease CH₂ FH₄ production (i.e., primary folatedeficiency, B12, B6, and other B-vitamin deficiencies which impairfolate one-carbon metabolism), or from antimetabolites drugs such as5-FU or methotrexate. Methotrexate inhibits dihydrofolate reductase,thus blocking the regeneration of tetrahydrofolates from dihydrofolate.5-FU and other fluorinated pyrimidines (for example, floxuridine, FUDRor trifluoromethylthymidine) block TS activity through formation of thespecific metabolite for this effect, fluorodeoxyuridylate (FdUMP),discussed below.

Inhibition of TS activity leads to "thymineless cell death" or"unbalanced cell growth," whereby RNA and protein synthesis, and cellenlargement, occur in the absence of adequate new DNA synthesis (seeGoulian et al., Adv. Exp. Med. Biol. 195:89-95 (1986), and refs.therein). In blood cells, such unbalanced cell growth can lead tomegaloblastic anemia, macrocytosis, and bone marrow failure.

The mechanism of inhibition of TS by FdUMP has been studied intensivelyfor the past two decades (see Santi et al., Biochem., pp. 8606-13,(1987) and refs. therein). In the absence of CH₂ FH₄, FdUMP binds TSextremely weakly. However, in the presence of a large excess of CH₂ FH₄,even low levels of FdUMP will bind tightly to TS, by forming inhibitoryTS-FdUMP-CH₂ FH₄ ternary complexes. In the presence of excess CH₂ FH₄,such ternary complexes are stable and no significant TS activity occurs.The molecular basis for the ternary complex is that after CH₂ FH₄ring-opening to form a covalent bond to FdUMP in the TS enzyme pocket(analogous to the normal reaction with deoxyuridylate), no hydride iontransfer can occur. Thus, no dihydrofolate is formed and thecovalently-bonded FdUMP-CH₂ FH₄ only leaves the enzyme site with greatdifficulty, as long as free CH₂ FH₄ is present in substantial excess. Ifthe CH₂ FH₄ concentration is relatively low, the ternary complexdissociates back to starting products, including free, active TS.

Thus, TS inhibition can occur with only trace amounts of FdUMP in slightexcess over TS molecules; however, a specific condition must occur inthat 5-10-methylenetetrahydrofolate (CH₂ FH₄) or tetrahydrofolate (FH₄)(and their polyglutamates) must be present in high concentration. Statedmore simply, CH₂ FH₄ is like a "glue" that holds the FdUMP onto the TSenzyme and therefore inhibits TS activity. However, CH₂ FH₄ is also apowerful growth factor, for promotion of purine, protein, and lipidmetabolism, as well as pyrimidine synthesis; thus, CH₂ FH₄administration for the purpose of promotion of TS inhibition by FdUMPmay be expected to also increase the degree of "unbalanced cell growth."

CH₂ FH₄ is a normal intracellular metabolite of the B-vitamin, folicacid, for use in thymidylate synthesis by TS. The same is true withrespect to the polyglutamates of CH₂ FH₄. However, CH₂ FH₄ is also usedby several other enzymes including CH₂ FH₄ reductase (EC 1.1.99.15),serine hydroxymethylase (EC 2.1.2.1), and Cl-tetrahydrofolate synthaseand CH₂ FH₄ dehydrogenase (EC 1.5.1.5). These interconversions using CH₂FH₄ are essential for purine synthesis, amino acid synthesis (i.e.,serine and methionine), and lipid metabolism through the re-methylationof methionine. Thus, CH₂ FH₄ is located at a metabolic branch point as asubstrate for at least 4 different enzymes (Green et al., Biochem.27:8014-22, (1988), S. J. Benkovic, Ann. Rev. Biochem. 49:227-51 (1980)and Schirch et al., Arch. Biochem. Biophys. 269:317-80 (1989)). Thisexplains the fact that intracellular CH₂ FH₄ is normally present in lowconcentrations, below 1.0 micromolar. Recent measurements have shownthat intracellular CH₂ FH₄ levels are typically low, and virtuallyalways lower than tetrahydrofolate, using the bacterial L. CaseiTS-[3H]FdUMP ligand binding assay (Priest et al., Cancer Res.48:3398-3404 (1988), and refs. therein). The present inventors havemodified this assay (Adv. Exp. Med. Biol. 244:98-104 (1988) and Invest.New Drugs 7:27-36 (1989)) and reported relatively low levels of CH₂ FH₄(much below 1.0 micromolar) in patients' cancer biopsy specimens despiteadministration of high doses of leucovorin (LV) (Proc. Am. Soc. Clin.Oncol. 8:69 (1989)). Furthermore, these observations of the presentinventors led to administration of the amino acid, L-serine, to patientsin an attempt to convert the tetrahydrofolates (in various polyglutamateforms, present in large excess) to CH₂ FH₄ (and polyglutamates). Theseresults have suggested that increased FH₄, rather than CH₂ FH₄, may betherapeutic. The inventors have recently published the only comparativedata that exist for the different major intracellular one-carbon formsof folates (Biochem. Pharmacol. 38:2985-93 (1989)), showing that of allof these, CH₂ FH₄ (at least, as the monoglutamate) is the best folateform for formation of TS-FdUMP-folate ternary complexes, and that aconcentration of CH₂ FH₄ in excess of 1.0 micromolar is desirable forthis effect. CH₂ FH₄ was found to be four times stronger than the nextbest folate, tetrahydrofolate, and about 100 times stronger than LV.However, to maintain CH₂ FH₄ as this form (vs. aqueous dissociation toFH₄), formaldeyde was required to be present in great excess over thefolate at these micromolar concentrations of folate.

Leucovorin (referred to as LV, or folinic acid) is(6R,S)-5-formyl-tetrahydrofolate and has been available commercially fordecades for the treatment of folic acid (the B-vitamin) deficiencystates (The Pharmacologic Basis of Therapeutics, 4th ed. (Goodman etal., eds.) The MacMillan Co., Toronto, pp. 1431-44 (1970)). In 1982, thefirst clinical reports of the usefulness of LV as a modulator of 5-FU incancer treatment appeared. (Machover et al., Cancer Treat. Rep.66:1803-07 (1982)). LV addition to 5-FU appeared to approximately doubleresponse rates in patients with gastrointestinal cancers. This resultwas confirmed in several subsequent studies. (For an extensive review,see Grem et al., Cancer Treat. Rep. 71:1249-64 (1987)). Currently, LVaddition to 5-FU therapy is community standard practice in the UnitedStates.

The mechanism of leucovorin (LV or folinic acid) improvement in theantitumor therapy of 5-FU and floxuridine (FUDR) has been shown inseveral studies to be due to improved TS inhibition associated withincreased intracellular (6R)-CH₂ FH₄ and (6S)-tetrahydrofolates.However, LV appears to be only partially effective in the goal ofpromoting complete TS inhibition by FdUMP in vivo. For an in vitroexample, researchers have shown that TS inhibition after 5-FU, whileimproved by LV, was still clearly incomplete (Keyomarsi et al., J. Biol.Chem. 263:14402-09 (1988)). In part, this may have been related tosaturation of obtainable summed pools of CH₂ FH₄ +FH₄ at about a 5-foldincrease over baseline at 30 hr LV exposure. Thus, maximum synergy of LVwas obtained at less than 1.0 micromolar exposure, with no furtherimprovement at higher concentrations although human plasma folates (LVand methyltetrahydrofolate, MTHF) are higher than this after high-doseLV administration (Doroshow et al., NCI Monogr, 5:171-74 (1987)). Arelated observation may be that addition of high-dose folic acid (140mg/m²) to 5-FU therapy appears to be associated with an increase intoxicity without improved response rates (Asbury et al, Am. J. Clin.Oncol. 10:47-49 (1987)).

In fact, decreasing synergy has been shown for LV addition to FUDR atconcentrations above 0.5 micromolar, when the colon cancer cells werepreviously folate-deficient (Davis et al., Mol. Pharmacol. 35:422-27(1989)). Also, others have shown in vivo in mice that expansion ofbreast tumor CH₂ FH₄ pools was a maximum of less than two-fold overbaseline despite massive LV dosing (180 mg/kg×8 over 48 hr) (Wright etal., Cancer Res. 49:2592-96 (1989)). These observations are mirrored inrecent clinical trials comparing the therapeutic outcome in coloncancer, in which low-dose LV (20 mg per square meter) was more effectivethan high-dose LV (200 mg per square meter) in terms of both tumorresponse rate and patient survival (Poon et al., J. Clin. Oncol.7:1407-18 (1989)). The lack of effectiveness of high-dose LV inpromoting complete TS inhibition was suggested by researchers based ontumor biopsy analyses in breast cancer patients: LV increased TSinhibition from an average of 30±13 to 71±14%, with responding patientsshowing the higher percentages of TS inhibition than non-responders(Swain et al., (J. Clin. Oncol. 7:890-99 (1989)).

In view of the above, the present inventors realized the potential ofthe direct administration of CH₂ FH₄ to patients receiving 5-FU, as sucha course of action would maximize TS inhibition.

The desirability and ability to use CH₂ FH₄ in the method of the presentinvention have never been obvious for various reasons.

For example, CH₂ FH₄ as a compound in solution has enjoyed a generalreputation of being extremely unstable. (Temple et al., "Chemical andPhysical Properties of Folic Acid and Reduced Derivatives," In Folatesand Pterins (Blakely et al., eds.), Vol. 1, pp. 61-63 (1984) and Wrightet al., Cancer Res. 49:2592-96 (1989)). In solution, it is generallyknown to exist in equilibrium with FH₄, requiring excess formaldehyde(CH₂ O) to favor the equilibrium toward CH₂ FH₄.

Under anaerobic conditions, such as made possible for clinicaladministration of CH₂ FH₄ or FH₄ by a closed, delivery system (U.S. Pat.No. 4,564,054), powdered FH₄ is stable even at room temperature, for ayear or more (Caldwell et al., Prep. Biochem. 3:323-26 (1973)).

Additionally, published data on the clinical tissue levels of CH₂ FH₄and FH₄ in patients have been limited, and it is well known that LV canbe given in gram-size doses (Grem, et al., supra.). LV is an extremelypowerful folate (B-vitamin) that is one-hundred times stronger thanfolic acid in correcting nutritional folate deficiency. As little as 1.0mg of LV will correct folate deficiency as a single dose (ThePharmacological Basis of Therapeutics, supra.). Thus, it is logical toassume that tumor CH₂ FH₄ and FH₄ levels might reach saturation levelsfrom high dose LV.

Finally, it appears that no published studies exist on the toxicologicalaspects of CH₂ FH₄ or FH₄. More specifically, there seems to be noavailable published work on either in vitro or in vivo effects of directexposure of living cells to CH₂ FH₄ or FH₄ except to rescue methotrexatetoxicity (Kisliuk et al., Cancer Treat. Rep. 61:647-50 (1977)).

Thus, in view of the structural properties of CH₂ FH₄ and FH₄ as well asthe lack of information regarding the biological effects of CH₂ FH₄ andFH₄, the present invention is quite remarkable. CH₂ FH₄ is utilized topotentiate or modulate the antitumor effects of the chemotherapeuticagent 5-FU.

L. R. Hughes (Eur. Pat. Appl. EP 284,3380 and Chem. Abstr. 110:95789(1989)) has described a novel folate analog as a TS inhibitor andantitumor agent. However, the discovery is clearly radically differentfrom the present invention. The analog does not occur naturally, isabsent two nitrogen atoms, is not reduced, and has a reactive propargylgroup attached to the glutamate moiety. Also, no mention is made of5-FU.

Interleukin-2 has been proposed as a modulator of tetrahydrobiopterin(U.S. Pat. No. 4,752,573); however, interleukin-2 is an oligopeptidehaving no resemblance to leucovorin, and no claim for TS inhibition orinteraction with 5-FU is made.

A patent for radiolabeled assay of folates (U.S. Pat. No. 4,136,159) hasno therapeutic pharmaceutical intent, and makes no mention of TSinhibition.

Various patents exist for other, unnatural folate analogs, includingquinazolines and dideazatetrahydrofolates as inhibitors of enzymes suchas folylpolyglutamyl synthetase (e.g., see Chem. Abstr. 110: P39366p(1989)). However, these are unnatural analogs which have distinctchemical, structural differences from CH₂ FH₄.

The European patent application (EP 266,042) of Wood et al. describes aprocess for separation of diastereomers of LV, as well as (6R)- and(6S)-tetrahydrofolates. No use of CH₂ FH₄ or FH₄ as a potentiator of TSinhibition by FdUMP (and thus 5-FU and other fluoropyrimidines) isclaimed in the document.

All U.S. patents and publications referred to herein are herebyincorporated by reference.

SUMMARY OF THE INVENTION

The present invention relates to the compound CH₂ FH₄ and its solutionproduct isomer FH₄, therapeutic uses of these compounds, andcompositions thereof. CH₂ FH₄ and FH₄ strongly potentiate the antitumoror TS-inhibitory effects of 5-FU.

More specifically, the present invention includes a method of inhibitingthe growth of a tumor in a patient comprising administering to saidpatient an amount of parent CH₂ FH₄ or FH₄ and 5-FU sufficient to effectsaid growth inhibition. The CH₂ FH₄ or FH₄ may be administeredconcurrently with 5-FU, or prior to the administration of 5-FU. In thelatter case, the CH₂ FH₄ or FH₄ is administered 6-24 hours, orpreferably 1-3 hours, before the administration of the 5-FU.

The CH₂ FH₄ or FH₄ may also be administered after the administration of5-FU in which case the CH₂ FH₄ or FH₄ compound is administered 1-10days, or preferably 1-6 hours, after the 5-FU administration.

Furthermore, the CH₂ FH₄ or FH₄ solution may be administered eitherintravenously, intraarterially, or intraperitoneally, and in a dosage of5-500 mg/m² (body surface area). Preferably, it may be administered in adosage of 20-200 mg/m² (body surface area). The CH₂ FH₄ or FH₄ solutionmay also be administered orally or topically as a 0.5% cream under anocclusive dressing.

If it is administered intravenously, such as through a central venouscatheter, the CH₂ FH₄ or FH₄ solution may be given in a dosage of 5-500mg/m² (body surface area), or preferably 20-200 mg/m², every 4-6 hours,once daily, or once weekly or as a continuous infusion of 20-200 mg/m²/week. Additionally, if it is administered every 4-6 hours, the CH₂ FH₄or FH₄ solution may be administered prior to, or subsequent to, theadministration of 5-FU.

The CH₂ FH₄ or FH₄ may be administered as the 6R, 6S, or as a mixture ofthe 6R and 6S enantiomers (diastereomers). Furthermore, one may varywhen to administer the compounds. For example, the natural (6S)diastereomer of FH₄ may administered 6-24 hours before administration of5-FU, or the natural (6S) diastereomer of FH₄ may administered 1-6 hoursafter administration of 5-FU. The natural (6S) diastereomer of FH₄ maybe administered concurrently with 5-FU. Similarly, the unnatural (6R)diastereomer of FH₄ may be administered 1-6 hours after theadministration of 5-FU, or the unnatural (6R) diastereomer of FH₄ may beadministered 6-24 hours after the administration of the natural isomerif the natural isomer is administered after the 5-FU.

Also, if the CH₂ FH₄ or FH₄, is administered in an alkaline vehicle, theconcentration of the CH₂ FH₄ or FH₄ is from 0.1 to 20 mg/ml whereas ifthe compound is administered in physiologic saline, the concentration isfrom 0.1 to 10 mg/ml.

Furthermore, the present invention includes a method of using CH₂ FH₄ orFH₄ in order reduce the toxicity of an anti-folate drug which has beenadministered to a patient. Examples of anti-folate drugs includemethotrexate, trimetrexate, nitrous oxide, and dideoxytetrahydrofolicacid.

The present invention also includes a method of treating folatedeficiency states by the administration of CH₂ FH₄ or FH₄.

Moreover, the present invention also includes a method of treating B12-and B6- refractory anemias whereby CH₂ FH₄ or FH₄ is administered in anamount sufficient to effect said treatment.

Furthermore, the present invention also includes a compositioncontaining CH₂ FH₄ or FH₄ and 5-FU, as well as a pharmaceutically activecarrier. The composition may also contain a stabilizing agent such as anascorbate salt, or glutathione. The composition may also contain freeformaldehyde. The FH₄ may be present in its natural (6S) diastereomericform or in its unnatural (6R) diastereomeric form.

Additionally, the present invention also includes a compositioncontaining CH₂ FH₄ or FH₄ and a compound which is metabolized to FdUMP,as well as a pharmaceutically active carrier. Examples of compoundswhich can be metabolized to FdUMP include floxuridine (FUDR), ftorafur(tegafur), and 5'-deoxyfluorouridine (Doxifluridine®). The compositionmay also contain a stabilizing agent, such as an ascorbate salt, orglutathione. Formaldehyde may also be present in the composition.

The present invention also encompasses a method of inhibiting the growthof a tumor in a patient, and inhibiting TS activity in the tumor,comprising administering to the patient an amount of unnaturaldiastereomer (6S)-CH₂ FH₄ or unnatural diastereomer (6R)-FH₄ sufficientto effect the growth and activity inhibition. The unnatural diastereomer(6S)-CH₂ FH₄ or unnatural diastereomer (6R)-FH₄ is administered in adosage of 5-500 mg/m², and preferably, in a dosage of 20-200 mg/m².

The natural diastereomer may be administered with the unnaturaldiastereomer. For example, the natural diastereomer (6S)-FH₄ and theunnatural diastereomer (6R)-FH₄ may be administered together in a ratioof from 1:20 to 20:1. The sum of the diastereomers is 5-500 mg.

The invention also includes a composition comprising (6R)-FH₄ and apharmaceutically acceptable carrier and a composition comprising(6S)-CH₂ FH₄ and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the effect of CH₂ FH₄ ("CH₂ H₄ PteGlu₁ ") on TSinhibition in 5-FU-resistant colon cancer cells (from tumor 51) afterthe administration of 5-FU ("FUra").

FIG. 2 represents the structure of (6R,S)-methylene-tetrahydrofolic acid(or CH₂ FH₄) and the configuration of the natural (6R)-CH₂ FH₄enantiomer (diastereomer) (Poe et al., Biochem. 18:5528 (1979) andKalbermatten et al., Helv. Chim. Acta 64:2633 (1981)).

FIG. 3 represents the structure of tetrahydrofolic acid or FH₄, thepredominant form at concentrations of less than 1 mM. The drawingrepresents either the (6R) or (6S) diastereomer.

FIG. 4 shows the results of TS-[³ H]FdUMP-folate binding assay of CH₂FH₄ as a function of concentration of the folate in 0.2M Tris buffer, pH7.4, with and without formaldehyde (CH₂ O), 6 mM, addition.

FIG. 5 shows the results of an experiment comparing the effects ofnatural (6R)-CH₂ FH₄ versus unnatural (6S)-CH₂ FH₄ on rats bearingtransplanted colon cancer. The filled circles are the weights andvolumes of tumors of rats treated with the unnatural (6S)-CH₂ FH₄diastereomer (mostly (6R)-FH₄ material). The triangles are results forthe natural (6R)-CH₂ FH₄ (mostly (6S)-FH₄), and the open circles,non-treated tumors. It should be emphasized that no fluorinatedpyrimidine was used, just the folate injections.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the present invention relates to the use of CH₂ FH₄ asa modulator of 5-FU in cancer chemotherapy. CH₂ FH₄ as well as FH₄,increase response rates to 5-FU as a result of increasing the inhibitionof TS by the 5-FU metabolite, FdUMP, in tumors. Thus, CH₂ FH₄ can beused to inhibit the growth of tumors when used in combination with 5-FU,or with other drugs which are metabolized to FdUMP including floxuridine(FUDR), ftorafur (tegafur), and Doxifluridine® (5'-deoxyfluorouridine).

The mechanism of action of CH₂ FH₄ is promotion of TS inhibition byFdUMP in fluoropyrimidine-treated tumors, which can occur by increasingthe rate of formation and stability of TS-FdUMP-CH₂ FH₄ and TS-FdUMP FH₄ternary complexes. The TS-FdUMP-FH₄ complexes are probably thetherapeutic objective for both the natural ((6S)-FH₄) and unnatural((6R)-FH₄) forms versus complexes with CH₂ FH₄. The blood and tumortissue levels of FH₄ are much higher than CH₂ FH₄. (6S)-FH₄ (especiallypolyglutamates) can form very strong ternary complexes without a risk of"fueling" residual TS activity as can occur with CH₂ FH₄, and at thesame time promote unbalanced growth. Furthermore, the unnatural (6R)-FH₄forms stronger (inhibitory) ternary complexes in the absence of excessformaldehyde. Administration of CH₂ FH₄ (as parent powder) in dosesranging from 5-500 mg/m² (body surface area), or preferably 20-200mg/m², will result in expansion of intracellular pools of FH₄ asmonoglutamates. One carbon exchange between endogenous CH₂ FH₄-polyglutamates and tetrahydofolatemonoglutamate resulting from CH₂ FH₄administration, as suggested in Tables II and III, would indicate thatthe optimal times for bolus 5-FU administration are concurrently or atseveral hours after bolus I.V. CH₂ FH₄ administration and thus aftermaximum polyglutamation. CH₂ FH₄ may also be administered after 5-FU isgiven or as a protracted, continuous infusion.

More specifically, CH₂ FH₄ may be administered 6-24 hours, orpreferably, 1-3 hours, prior to the administration of 5-FU. CH₂ FH₄ canalso be administered 1-10 days, or preferably 1-6 hours, subsequent tothe administration of 5-FU.

Polyglutamation of folates causes retention within the cell, andtypically also accelerates rates of enzyme processing of one-carboninterconversions of folates (Schirch et al., Arch. Biochem. Biophys.269:371-80 (1989), Green et al., Biochem. 27:8014-22, 1988). Currentdata would suggest that polyglutamation of FH₄ and CH₂ FH₄ will promoteTS-FdUMP-folate inhibitory ternary complex formation to a greater extentthan promotion of the normal enzymic reaction with deoxyuridylate(Houghton et al., Cancer Res. 48:3062-69 (1988)). Since polyglutamatesmay form TS-FdUMP-folate ternary complexes as much as 50-fold moretightly than parent monoglutamates, an objective of folate addition tofluoropyrimidine therapy could also include formation ofTS-FdUMP-tetrahydrofolates, which would also be strongly inhibitory. Inaddition, a role for the unnatural enantiomers (diastereomers at thepterin C6- position), such as polyglutamates of (6S)-CH₂ FH₄ or(6R)-FH₄, in TS inhibition by forming TS-deoxyuridylate-folate orTS-FdUMP-folate ternary complexes, potentially could be a factor(Kisliuk et al., Biochem. 20:929-34 (1981)) in the TS inhibitionobserved with CH₂ FH₄ administration in vivo (Tables I, II, and III;FIG. 1).

The potentiation of TS inhibition by low levels of FdUMP may be expectedto last only a few hours unless polyglutamation of the CH₂ FH₄ and FH₄occurs thereby creating more powerful TS-FdUMP binders than the parentmonoglutamate. Thus, CH₂ FH₄ dosing requirements may be as frequent asevery 4-6 hrs., once daily, or as infrequent as once weekly.

In one embodiment of the present invention, CH₂ FH₄ can be administeredby intermittent (e.g., daily) bolus dosing in patients who have centralvenous catheters. Such patients could self-administer the CH₂ FH₄ (usinga means for ensuring the stability of the formulation to oxidation) andwould also be candidates for administration of CH₂ FH₄ by continuous,intravenous protracted infusion. The 5-FU infusion would be expected toproduce low levels of FdUMP in tumors. Low FdUMP levels would beexpected to be associated with relatively poor TS inhibition unless CH₂FH₄ levels were very high. FH₄, free of formaldehyde as a stabilizer mayalso be administered in the same manner.

An ameliorating factor to consider may be that chronic TS inhibition,albeit incomplete, would be expected to cause slight increases in CH₂FH₄ levels because of lowered consumption of CH₂ FH₄ in the natural TSmechanism so that pharmaceutical CH₂ FH₄ in this setting might be moreefficient.

Other embodiments include the addition of CH₂ FH₄ at late times afterbolus intravenous 5-FU infusion (e.g., at 6 hours in the daily 25(monthly) Schedule, or at days 4, 5 and 6 on the biweekly bolusschedule.)

In addition to being administered intravenously, CH₂ FH₄ may also beadministered intraarterially or intraperitoneally, also in a dosage of5-500 mg/m², or preferably, in a dosage of 20-200 mg/m². However, CH₂FH₄ may also be administered topically as a 0.5% cream under anocclusive dressing.

Another embodiment of the present invention comprises a compositioncontaining CH₂ FH₄ (or FH₄ in solution) as well as 5-FU. The compositionalso contains a pharmaceutically active carrier, and may also containformaldehyde in excess as a stabilizer against oxidation.

A further embodiment of the present invention includes a compositioncontaining CH₂ FH₄ and one or more other drugs which can be metabolizedto FdUMP. The composition may contain a pharmaceutically active carrier,and may also contain formaldehyde in excess as a stabilizer.

It should be noted that FH₄, free of formaldehyde, can replace the useof CH₂ FH₄ in each of the above embodiments. Each of the two compoundshas a natural and unnatural diastereomeric form (i.e., natural (6R)-CH₂FH₄, unnatural (6S)-CH₂ FH₄, natural (6S)-FH₄ and unnatural (6R)-FH₄).Each form may be administered before, after, or concurrently with 5-FU.Furthermore, the unnatural forms may be used alone in a dosage of 5-500mg/m², without fluoropyrimidines, as agents for TS inhibition andinhibition of tumor growth. Moreover, different ratios of (6S)-FH₄ and(6R)-FH₄ together may be utilized in treatment if provided in a 1:20 to20:1 ratio (5-500 mg/m² sum).

It should also be noted that the different diastereomers may be given atdifferent times to the same patient. For example, the natural (6S)-FH₄(solution of (6R)-CH₂ FH₄) may be administered several minutes or hoursbefore or after (or concurrently with) 5-FU administration. Theunnatural (6R)-FH₄ (solution of (6S)-CH₂ FH₄ powder) may then beadministered several hours later, at a time when the cancer cells arerecovering from unbalanced growth and new TS activity is occurring.

Because reduced folates are rapidly interconvertible according to theirone-carbon states, it may be anticipated that the clinical tolerance forCH₂ FH₄ or FH₄ will be similar to that of LV (6R,S-leucovorin) and5-methyl-(6R, S)-tetrahydrofolate (MTHF), the natural diastereomer ofthe latter being the predominant blood transport form of folates.

Also, FH₄, and possibly CH₂ FH₄, have recently been reported asaccumulating to low but significant (i.e., less than 20 micromolar)concentrations in human plasma after LV administration to human subjects(Bunni et al., Cancer Chemother. Pharmacol. 23:353-57 (1989)).

Thus, it can be anticipated that the dose tolerance for CH₂ FH₄ or FH₄in humans is similar to the reported experiences with LV andmethyltetrahydrofolate (MTHF) (both of which are given as a mixtures ofenantiomers (diastereomers)). Specifically, an upper limit of 500 mg persquare meter body surface area would be expected to be therapeuticallyeffective. The lowest effective dose may possibly be more powerful thaneither LV or MTHF, and thus could be as low as 5 mg per square meterbody surface area in a single dose. A dosage of 20-200 mg/m² (bodysurface area) is preferred.

Based on previous studies of the toxicology of folates (LV, MTHF andfolic acid) combined with 5-FU and fluorodeoxyuridine, the LD50 in ratswould be expected to be above 150 mg/kg i.v. (single bolus) with regardto CH₂ FH₄ or FH₄, and may be expected to cause convulsions in such highdoses (Bartosek et al., Chemioterapia Oncologica 2(4): 85-98 (Dec. Supp,1987)).

The pH of the CH₂ FH₄ /FH₄ solution which is to be injected, may rangefrom slightly acidic to slightly alkaline. 5-FU up to 50 mg/mL inalkaline media may be present, analogous to the practice of formulationof 5-FU and LV in the same solution (e.g., Trave et al., J. Clin. Oncol.6:1184-91 (1988)). Furthermore, the concentration for injection may beas high as 100 mg/10 mL, preferably from 0.1 to 20 mg/ml, in alkalinevehicles. The concentration may also be as high as 100 mg/20 mL,preferably from 0.1 to 10 mg/ml, in physiologic, normal saline. Atconcentrations less than 1 mM in initial CH₂ FH₄ concentrations, thepredominant form in solution is FH₄ (i.e., the dilution of CH₂ FH₄ inaqueous solution shifts the equilibrium between FH₄ and CH₂ FH₄ towardsFH₄, regardless of pH, O₂ tension, or the presence of reducing agents).

Ascorbate salts may be present as stabilizers (e.g., 1% w/v as the saltat neutral or slightly alkaline pH). Other reducing substances may alsobe used as stabilizers, for example, reduced glutathione.

Free formaldehyde (CH₂ O) may also be present in concentrations up to 10mM. However, the dosage must be adjusted for formaldehyde toxicity. Theformulation may be made directly from (6R,S)-FH₄ powder, alternatively.In this case, formulations would be checked and controlled for thedegree of spontaneous condensation of formaldehyde as a contaminant fromambient air to form CH₂ FH₄ (Pogolotti et al., Biochem. 18:2794-2804(1979)). The oral LDLo (or lowest lethal dose) of CH₂ O in humans hasbeen reported to be 36 mg/kg (Registry of Toxic Effects of ChemicalSubstances, US DHHS, PHS, CDC, NIOSH, Vol. 1, p. 822 (1980)). The pure(6R)CH₂ FH₄ or (6S)FH₄ enantiomer may also be utilized, free of theunnatural (6S)CH₂ FH₄ or (6R)FH₄ enantiomer, respectively. Enantiomer(diastereomer) separation is obtainable by chiral column or DEAE columnpreparative isolation (Kaufman et al., J. Biol. Chem. 238:1498-1500(1963)).

A possible major advantage of CH₂ FH₄ over FH₄ as the parent powderedmaterial is the protection against air oxidation, referred to above,which protection would therefore be greater with concentrated versusdilute (e.g., <0.5 mM) concentration, in the absence of a mechanism forexcluding air during reconstitution and administration (as provided bythe Protector device).

It appears that direct administration of CH₂ FH₄ or FH₄, either as themixture of 6R and 6S diastereomers (enantiomers), the unnatural 6S-CH₂FH₄, or the natural 6R-CH₂ FH₄ alone (or their FH₄ solution equilibriumproducts, referred to as solution product isomers (see pages 1 and 10))can overcome some of the disadvantages of LV described above. That is,CH₂ FH₄ addition to 5-FU can lead to greater tetrahydrofolate (FH₄) andCH₂ FH₄ elevations intracellularly than LV or MTHF (which both requireone carbon activation), and consequently show more profound synergism onTS inhibition by FdUMP.

The applications for CH₂ FH₄ or FH₄ are quite significant andfar-reaching. For example, antitumor uses of CH₂ FH₄ or FH₄, combinedwith TS-inhibitory fluoropyrimidines include: 1) addition toPlatinol/5-FU infusion therapy in head and neck cancer and otherepidermoid cancers, 2) addition to combinationcyclophosphamide/doxorubicin/5-FU in breast cancer 3) addition totopical Efudex® (5-FU) cream under an air-free occlusive dressing forskin conditions (for example benign keratoses, keratoacanthomas,verrucae, premalignant keratoses, in situ cancer and invasivesuperficial malignancies amenable to topical therapy). Furthermore, CH₂FH₄ or FH₄ can also be applied to those cancer types in which 5-FU andfloxuridine are typically combined with LV, such as in colon, rectal andpancreatic carcinomas.

CH₂ FH₄ or FH₄ can also be utilized with respect to non-malignancyrelated conditions. For example, the natural diastereomers of CH₂ FH₄ orFH₄ can be used with respect to B12- and B6-refractory anemias which arenot responsive to LV. CH₂ FH₄ or FH₄ can also be used to treat folatedeficiencies. Furthermore, CH₂ FH₄ and FH₄ can also be used for thepotentiation (selective rescue of the host patient) of the TS inhibitorymechanism of antibacterial action of nucleotide analogs.

Additionally, CH₂ FH₄ or FH₄ can be utilized to reduce the toxicity ofanti-folate drugs which have been administered to patients. Suchanti-folate drugs include, for example, methotrexate, trimetrexate,nitrous oxide, and dideoxytetrahydrofolic acid.

As a rescue agent following methotrexate, CH₂ FH₄ or FH₄ may be morespecific than the presently used LV (or MTHF) since CH₂ FH₄ wouldrequire less (or no) metabolic activation in the case of FH₄ to providefor purine, pyrimidine, and the amino acid synthetic requirementsnormally met by intracellular folates. CH₂ FH₄ could also thereforebecome useful in rescue of the host in the trimetrexate treatment ofPneumocystis carinii infections of immunosuppressed patients (i.e., AIDSpatients).

The present invention can be illustrated by the use of the followingnon-limiting examples.

EXAMPLE 1 Synthesis of CH₂ FH₄ as a Low-Formaldehyde Material

Preparation of (6R,S)-CH₂ FH₄

CH₂ FH₄ as the equal mixture of diastereomers (optical isomers orenantiomers at the C6-position; both diastereomers are of the naturalL-configuration at the alpha-carbon position of the glutamate moiety)was prepared from (6R,S)-tetrahydrofolic acid, commercially availablefrom Sigma, in the examples described below. The method of synthesis hasbeen described previously (C. P. Spears and B. Gustavsson, Adv. Exp.Med. Biol. 244:98-104 (1988)). To (6R, S)-tetrahydrofolate (6R, S-FH₄)powder, (100 mg) is added 360 μL of 1.0M Na Ascorbate, pH 6.5, 68 μL of37% (w/w) formaldehyde (CH₂ O), and 16 mL phosphate buffer, pH 7.0. A10-min room temperature incubation allows completion of formation of(6R,S)-CH₂ FH₄. This material is applied to a DEAE-cellulose columnusing a modification of a well-known procedure (Kaufman et al., J. Biol.Chem. 238:1498-1500 (1963)). A step elution with NH₄ HCO₃ buffers ofincreasing concentration and pH, leads to isolation of CH₂ FH₄ in thelast pooled fraction. This material does not contain free formaldehydeas assayed colorimetrically, or by toluene extraction of dimedone(methone)-trapped [11-¹⁴ C] CH₂ FH₄, prepared with [¹⁴ C]CH₂ O asdescribed previously (Moran et al. Proc. Natl. Acad. Sci. USA76:1456-60, 1979). Phosphate buffers and TEAE-cellulose can also be usedin the procedure of Kaufman, which gives both enantiomers of CH₂ FH₄ inthe same peak; however, if potassium bicarbonate buffer is used, aseparation of the enantiomers is effected, with the biologically active,natural-configuration, (6R)-CH₂ FH₄ peak eluting after the (6S)-CH₂ FH₄peak. The amount of formaldehyde (as methylene) in the product may, infact, be even less than stoichiometric with tetrahydrofolate (FH₄)(Horwitz et al, J. Med. Chem. 12:49-51 (1969)). The amount of (6R)-CH₂FH₄ in the preparations is checked by one or more of the three followingmethods. (1) Spectrophotometrically, by use of this material as thelimiting substrate in a TS assay with L. Casei enzyme, as described byDaron et al. (J. Biol. Chem. 253:940-45 (1978); (2) ligand binding assayusing [6-3H]FdUMP and L. Casei TS described by the inventors (Adv. Exp.Med. Biol. 244:98-104, 1988); and by absorbance at 294 nm on HPLC (Lu etal., Biochem. 23:6870-75 (1984)). Column-isolated CH₂ FH₄, whetherracemic in 6R- and 6S-forms or as the 6R-form alone in solution can bestored under argon at -80° C. for up to a year without decomposition(Bruice, et al. Biochem. 21: 6703-09 (1982)). Alternatively, solutionsof CH₂ FH₄ after column isolation can be lyophilized to powder andstored under nitrogen in sealed glass ampoules. Various ratios offormaldehyde to CH₂ FH₄ can be used, from less than stoichiometric, asdescribed above, including no formaldehyde (either bound as methylene,or free) to a 2- to 4-fold or more excess (Bruice, et al., Biochem.21:6703-07, (1982)). The use of 2-mercaptoethanol or other reducedthiols has been advocated by some workers, but is unnecessary and maycause minimal interference (S. F. Zakrewski, J. Biol. Chem. 241:2957-961(1966) and Kallen et al. J. Biol. Chem. 241:5845-50 (1966)) incondensation of CH₂ O with tetrahydrofolate.

Alternative methods for synthesis and purification of (6R,S)-CH₂ FH₄ arereviewed by C. Temple, Jr. and J. A. Montgomery, In: Folates and Pterins(R. L. Blakley and S. J. Benkovic, eds.), vol. 1, Chemistry andBiochemistry of Folates, John Wiley & Sons, New York, pp. 61-120 (1984).This includes use of (6R,S)5-formyltetrahydrofolate (leucovorin or LV),which is commercially available in bulk quantities, and is converted tothe 5,10-methenyltetrahydrofolate by acidic conditions. The lattercompound then can yield CH₂ FH₄ by reduction with borohydride in DMSOand pyridine (Farina et al., J. Am. Chem. Soc. 95: 5409 (1973)).

Preparation of (6R)-CH₂ FH₄

The naturally-occurring diastereomer (enantiomer) of CH₂ FH₄, (6R)-CH₂FH₄, can be prepared by a number of methods, including that of Kaufmanet al. as described in the foregoing section, using TEAE-celluloseelution by bicarbonate. Commercially-available folic acid reduced todihydrofolate using hydrosulfite (Mathews et al. J. Biol. Chem.235:3304-08, (1960)) or dithionite (R. L. Blakley, Nature 188:231-32,(1960)) is used as a substrate for purified dihydrofolate reductase inthe present of NADPH (e.g., see M. Poe et al, Biochem. 18:5527-30(1979)). Formation of (6S)-tetrahydrofolate (which is the naturaldiastereomer) is readily followed at 294 nm. Purification is then doneby chromatography (e.g., S. F. Zakrewski and A. M. Sansone, MethodsEnzymol. 18B:728-31, 1971), followed by lyophilization to powder andstorage under nitrogen or argon in sealed glass vials.

An additional approach is reduction of dihydrofolic acid bydihydrofolate reductase in the presence of formaldehyde (Horne et al.,Methods Enzymol. 66:545ff (1980)), followed by column isolation, whichavoids the need for a separate CH₂ O step after (6S)-tetrahydrofolateisolation. In these preparations, ascorbate is typically present (e.g.,0.1M) as an antioxidant. Synthesis of the unnatural (6S)-CH₂ FH₄ isomerhas been described, by selective enzymic conversion of (6R)-CH₂ FH₄ todihydrofolate, which is easily separated by column chromatography (Anal.Biochem., Vol. 154, pp 516-24 (1986)). The isomeric solution of (6S)-FH₄is then obtained by dilution to less than 0.5 mM.

Stability of CH₂ FH₄

Solutions of CH₂ FH₄, as well as the powder, are unstable in thepresence of oxygen, with oxygen degradation being catalyzed by light,acid, base, and heavy metals (R. G. Kallen, Methods Enzymol. 183:705ff,1971). CH₂ FH₄ is somewhat more stable than FH₄, as are the majorN5-substituted tetrahydrofolates; FH₄ solutions can undergo 90%degradation in 4.1 hr when exposed to air (discussed in C. Temple, Jr.,and J. A. Montgomery, supra. However, tetrahydrofolate (FH₄) iscompletely stable under anaerobic conditions Caldwell et al., Prep.Biochem. 3:323-26 (1973).

Thus, a method for air-free reconstruction of CH₂ FH₄ or FH₄ powder (invacuum, or under nitrogen or argon in air-tight ampoules), or freshhandling of column-isolated CH₂ FH₄ or FH₄, is required to ensure thestability of CH₂ FH₄ as a pharmaceutical with accurate dosing. Theinvention of Gustavsson, one of the present inventors, (U.S. Pat. No.4,564,054) referred to as the Protector device, affords such a method.The Protector invention is not generally known, since it is marketed asa method for prevention of aerosolization of mutagenic/toxic cancerchemotherapy agents, however, it is equally useful for air-freereconstitution, dosing, and i.v. administration of drug solutions topatients. The Protector is suitable for handling all anticipated doseranges and concentrations of CH₂ FH₄, with the volume for dosing limitedonly by the syringe size. Vehicles for reconstitution of CH₂ FH₄ or FH₄powder include 5% dextrose, normal (0.89% w/v) saline, 5-FU solutions,and sterile water, (which may or may not be de-aerated for removal ofdissolved oxygen prior to use in reconstitution of CH₂ FH₄ or FH₄powder, depending on the presence in the formulation of antioxidantstabilizers such as ascorbate). The Protector may be modified to usesemi-opaque materials, such as brown plastic, to reduce transmission ofambient light.

EXAMPLE 2 CH₂ FH₄ Use With 5-FU in Murine Colon Carcinoma CA51

(6R, S)-CH₂ FH₄ was prepared by the DEAE-cellulose column procedure,described above, using step-elution of the material as previouslyreported for purification of nucleotides (Moran et al., Proc. Natl. Aca.Sci. USA 76:1456-60 (1979)). To twenty micromoles of (6R,S)-FH₄ (Sigma)were added 62.5 ul of 1.0M Na Ascorbate, pH 6.5, 2.7 ul of 37%formaldehyde stock, and 0.6 mL of 5 mM phosphate buffer, pH 7.0. Becauseof the high formaldehyde, this solution was over 2 mM in CH₂ FH₄, withless FH₄ present as the solution isomer. After 20 min at roomtemperature, this solution was applied to a 1×3-cm DEAE-cellulosecolumn; in the last step, the 500 mM NH4HCO3 (pH 8.0) fraction (30 mL)was pooled, lyophilized to dryness, and stored under vacuum in glassampoules. This fraction contains both diastereomers of CH₂ FH₄.Spectrophotometric assay (which uses mM concentrations of folate and CH₂O) of powder reconstituted in phosphate-buffered-saline showed aconcentration of the natural (6R)-CH₂ FH₄ in this solution of 2.4 mM;prior assay by L. casei TS-3H]FdUMP-folate ternary complex formationgave a concentration of 2.5 mM.

On the day of reconstituting the above CH₂ FH₄, mice bearingsubcutaneous murine colon carcinoma Tumor 51 were administeredintraperitoneal (i.p.) 5-FU, with or without concomitant i.p. CH₂ FH₄ byseparate injection. The 5-FU was given at a dose of 1.6 mg per mouse,about 80 mg/kg. The CH₂ FH₄ was given at a dose of 0.5 mL of the 2.4 mMmaterial (1.2 mmole/mouse), above. The in vivo methodologies wereessentially as had previously been described (C. P. Spears, et al.,Cancer Res. 42:450-56 (1982)). In contrast, however, to the extensiveprior experience of the present inventors with this 5-FU-resistant tumorline, which always had shown significant FdUMP-titratable free TSlevels, the tumors of mice receiving concomitant CH₂ FH₄ showedabrogation of TS activity (Table I and FIG. 1). The free TS levels ofthe 5-FU-only treated mice were comparable to the previous observationsof the inventors in this line, and at the 1.0 pmol/g level of TSactivity was sufficient to support thymidylate synthesis required fortumor growth (C. P. Spears, Exerpta. Med. Int. Congr. Series 647:12-19,(1984)). The levels of apparent free TS in tumors of mice receiving CH₂FH₄ concomitant with 5-FU were at, or below, that level due toexchange-labeling of endogenous TS-FdUMP-folate ternary complexes in thecytosolic extracts. Stated otherwise, the average ± S. D. apparent TSvalue of 0.42±0.20 pmol/g for the 5 tumors of the 5-FU+CH₂ FH₄ treatmentgroup when corrected downward for labeling of endogenous FdUMP-inhibitedenzyme by a minimum correction factor of 5% (Spears and Gustavsson, Adv.Exp. Med. Biol. 244:98-104, (1988)) equates with zero detectable TSactivity. This is exactly the qualitative difference between sensitivityand resistance to 5-FU previously established (see Spears et al., CancerRes. 42:450-52 (1982)). An additional observation was that in the Tumor51 specimens from mice receiving CH₂ FH₄ concomitant with 5-FU was thatthe pre-incubation dissociation condition, which had previously beenroutinely used for regenerating all TS in the free form, was completelyunable to regenerate free TS, in contrast to the more normal findings inthe 5-FU-only exposed tumors. This is strongly suggestive that CH₂ FH₄administration raised concentrations of tumor CH₂ FH₄ and FH₄, so high,that even after large dilution into the assays the concentrations werestill above those that could spontaneously oxidize to lower levelspermitting in vitro ternary complex dissociation.

The results obtained from Example 2 are shown in FIG. 1, and in Table I.

                  TABLE I                                                         ______________________________________                                        TS INHIBITION IN MURINE TUMOR CA51 AFTER 5-FU.sup.a                           EFFECT OF CO-ADMINISTRATION OF CH.sub.2 FH.sub.4 .sup.b                       (Values = Ave. ± S.D.)                                                     5-FU Alone         5-FU + CH.sub.2 FH.sub.4                                         Free TS.sup.c        Free TS.sup.c                                      Hours (pmol/g)  % Inhibition                                                                             (pmol/g)                                                                              % Inhibition                               ______________________________________                                        1     1.67      83.3       0.41    95.9                                             ±0.28  ±2.8    ±0.26                                                                              ±2.6                                                               0.164   98.4                                                                  ±0.13                                                                              ±1.3                                    3     1.00      90.0       0.36    96.4                                             ±0.72  ±7.2    ±0.06                                                                      0.71    92.9                                                                  ±0.03                                           6     1.27      87.3       0.46    95.4                                             ±0.06  ±0.6    ±0.05                                           ______________________________________                                         .sup.a 80 mg/kg i/p.                                                          .sup.b 27 mg/kg in (6R) CH.sub.2 FH.sub.4 by spectrophometric and binding     assays.                                                                       .sup.c Not corrected for ternary complex exchange labeling or ratio of        CH.sub.2 FH.sub.4 to FH.sub.4. A minimal correction factor of 5% leads to     the calculation that there was 100% TS inhibition for all tumors receivin     the combination of 5FU and CH.sub.2 FH.sub.4, compared to only 92% averag     TS inhibition by 5FU alone. Baseline total TS was 10.00 ± 0.04 pmol/g.

EXAMPLE 3 Effect of CH₂ FH₄ on Two Patients Who Had Already ReceivedTreatment With 5-FU

CH₂ FH₄ was formulated, assayed, and administered to 2 patients who hadpreviously been treated with 5-FU. The assays were performed by themethods described in Spears et al., Adv. Exp. Med. Biol. 244:98-104(1988). In the data shown, the TS inhibition profiles that resulted fromCH₂ FH₄ administration were not due to concurrent 5-FU dosing. The mostrecent exposure to 5-FU in these cases was slightly greater than a weekprior to the study date, with the patients eligible, however, from thestandpoint of toxicity evaluation to receive the weekly dose of 5-FU.Thus, residual FdUMP levels from previous exposure, below the detectablelimits for assay, were expected to be present (See spears et al. Mol.Pharmacol. 27:302-07 (1985)). The serial biopsies were done followingsingle dose administration of CH₂ FH₄.

The formulation of CH₂ FH₄ was as described in Example 2, and wasperformed on the day of CH₂ FH₄ administration. The assays were alsoperformed on the day of CH₂ FH₄ administration.

The results in these patients of the pharmacodynamic tumor tissueanalyses showed striking evidence of TS inhibition following CH₂ FH₄administration. These results are summarized in Tables II and III below.

                  TABLE II                                                        ______________________________________                                        TS INHIBITION AFTER CH.sub.2 FH.sub.4 ADMINISTRATION                          PATIENT:    A.M.; last 5-FU treatment: ≧1 week                         LOCATION:   Ostra Sjukhuset (Eastern Hospital), Sweden                        TUMOR:      Skin metastasis from gastric carcinoma                            CH.sub.2 FH.sub.4                                                                         0.1M Na Ascorbate, pH <9.5, Sigma                                 FORMULATION:                                                                              (6R,S)CH.sub.2 FH.sub.4, DEAE-column purified                     CH.sub.2 FH.sub.4 DOSE:                                                                   30 mg in 30 cc IV over 2 min; 4 mg                                            as parent CH.sub.2 FH.sub.4, 26 mg as FH.sub.4.                   (Tumor Tissue Values = Ave. ± S.D.)                                                 THYMIDYLATE                                                          Time     SYNTHASE (TS).sup.b                                                                           FBC.sup.c                                            of Biopsy.sup.a                                                                        pmol/g   % of Baseline                                                                            nmol/g % of Baseline                             ______________________________________                                         0 min   1.31     (100)      5.88   (100)                                              ±0.13            +0.56                                            10 min   0.26     19.8       0.23   3.9                                                ±0.17            +0.02                                            20 min   0.56     42.7       0.27   4.6                                                ±0.06            +0.01                                            40 min   0.99     75.6       0.21   3.6                                                ±0.08                                                             60 min   1.47     112.2      0.14   2.3                                                ±0.13            ±0.01                                         ______________________________________                                         .sup. a Biopsies of solitary skin metastasis, average weight 68 ± 58       mg, time after CH.sub.2 FH.sub.4 administration.                              .sup.b By [6-.sup.3 H]FdUMP ligandbinding assay (CP Spears et al., Cancer     Res. 42:450-56 (1982).                                                        .sup.c Folate Binding Capacity, FBC, is a measure of tissue CH.sub.2          FH.sub.4 and FH.sub.4 level (Invest. New Drugs 7:27-36 (1989), (modified      after Priest et al., Biochem. J. 216:295-98 (1983)), with a Sigma             (6R,S)CH.sub.2 FH.sub.4 standard value of 936 DPM/pmole.                 

                                      TABLE III                                   __________________________________________________________________________    TS INHIBITION AFTER CH.sub.2 FH.sub.4 ADMINISTRATION                          PATIENT:       K.H.; last 5-FU treatment: ≧1 week                      LOCATION:      Ostra Sjukhuset (Eastern Hospital), Sweden                     TUMOR:         Rectal adenocarcinoma, locally advanced                        CH.sub.2 FH.sub.4                                                                            0.2M Na Ascorbate, Sigma (6R,S)-CH.sub.2 FH.sub.4              FORMULATION.sup.a :                                                           CH.sub.2 FH.sub.4 DOSE:                                                                      35 mg IV over 1 min week #1; 50 mg IV in                                      40 ml week #2                                                  (Tumor Tissue Values = Ave. ± S.D.)                                        THYMIDYLATE                                                                   SYNTHASE (TS).sup.c   FBC.sup.d                                               Time  pmol/g  % of Baseline                                                                         ΔDPM                                                                            % of Baseline                                   of Biopsy.sup.b                                                                     Week #1 Week #2 Week #1 Week #2                                         __________________________________________________________________________     0 min                                                                              5.77                                                                              (100)                                                                             5.64                                                                              (100)                                                                             759 (100)                                                                             499 (100)                                             ±0.09                                                                              ±1.26                                                                              ±145 ±190                                         10 min                                                                              6.28                                                                              (212.4)                                                                           10.25                                                                             (181.7)                                                                           320 (42.2)                                                                            376 (75.4)                                            ±1.92                                                                              ±0.82                                                                              ±60  ±17                                          20 min                                                                              2.26                                                                              (43.7)                                                                            5.91                                                                              (104.8)                                                                           314 (41.4)                                                                            814 (163.1)                                           ±0.36                                                                              ±0.17                                                                              ±9                                                   30 min                                                                              5.90                                                                              (114.1)                                                                           2.02                                                                              (35.8)                                                                            632 (83.3)                                                                            249 (49.9)                                            ±0.12                                                                              ±0.03                                                                              ±26  ±75                                          40 min        3.46                                                                              (61.3)      399 (80.0)                                                    ±0.28        ±44                                          24 hr 6.32                                                                              (122.2)     1403                                                                              (184.8)                                                   ±0.52        +130                                                    __________________________________________________________________________     .sup.a On Week #1 the CH.sub.2 FH.sub.4 was formulated at pH 2.0,             DEAEpurified; On Week #2 the preparation was pH 9.0, with 6 mM (final         concentration) CH.sub.2 O added, no DEAE step used.                           .sup.b Biopsies of rectal pouch mass, average weights, 145 ± 39 mg         (Week #1) and 136 ± 24 mg (Week #2). Time after CH.sub.2 FH.sub.4          administration.                                                               .sup.c By [6.sup.3 H]FdUMP ligandbinding assay (Spears et al., Cancer Res     42:450-56 (1982)).                                                            .sup.d Folate Binding Capacity, given in ΔDPM over [.sup.3 H]FdUMPT     binary complex background (Invest. New Drugs 7:27-36 (1989)); standard        curve Sigma (6R,S)CH.sub.2 FH.sub.4 showed 920 and 898 ΔDPM/pmole       for weeks 1 and 2. Multiply ΔDPM values by 0.0002 to convert to         nmol/g.                                                                  

In patient A. M., a sixty-seven year old woman with over a 3 year priorhistory of disseminated gastric cancer, and who was end-stage in hercourse, TS was inhibited 80.1 and 57.3% in her tumor at 10 and 20 min,respectively, in her tumor after CH₂ FH₄ administration. (It should benoted that the CH₂ FH₄ preparation was over 85% FH₄.) Notably, when shewas studied again 2 weeks subsequently, with a repeat dose of CH₂ FH₄,TS in the baseline tumor biopsy was undetectable (data not shown).

The FBC (folate binding capacity of L. casei TS-[3H]FdUMP added to thecytosols, (a measure of tissue CH₂ FH₄ and FH₄, mostly presumed to bepolyglutamates) also showed a surprising decrease, which continuedthrough 60 min. Tissue FH₄ polyglutamates were not separately measuredby use of CH20 addition to the FBC conditions. The continuing drop inFBC, however, at the 60-min time point rules out the possibility thatall post-CH₂ FH₄ biopsies were somehow an artifact of tumor tissuesampling. This paradoxical decrease in FBC is a characteristic featureof 5-FU-responding patients receiving high-dose LV added to 5-FU bolusi.v. therapy (C. P. Spears, et al. Presentation at 25th Annual Am. Soc.Clin. Oncol. meeting, May 22, 1989). This decrease was also seen intumor of patient K. H. (Table 3). An explanation for the paradoxicaldecrease in FBC is that one-carbon exchange (e.g., R. G. Matthews et al,Adv. Enz. Regul. 26:157-70 (1987) occurred in the tumor tissue, betweenFH.sub. 4 -monoglutamate derived within minutes from administration ofthe CH₂ FH₄ /FH₄ drug, and endogenous CH₂ FH₄ -polyglutamates. Since thepolyglutamates of CH₂ FH₄ may be expected to bind TS-FdUMP up to 50-foldmore strongly than the monoglutamate (Houghton et al., Cancer Res.48:3062-69 (1988)), the one-carbon exchange could lead to the observeddecrease. This data is powerful evidence that CH₂ FH₄ /FH₄ given to thispatient was rapidly transported and metabolized in her tumor. Thedecrease in TS in her tumor, then, is assumed to be related to thismetabolism and the presence of non-measurable levels of FdUMP (atconcentrations near stoichiometry with endogenous TS binding sites). Theparadox of decreasing free TS with decreasing FBC also can be explainedby metabolic channeling of administered CH₂ FH₄ (Reddy et al., Proc.Natl. Acad. Sci. USA 77:3312-16, 1980), or by formation ofTS-FdUMP-tetrahydrofolate, or of TS-deoxyuridylate-CH₂ FH₄ ternarycomplexes by the unnatural (6S)-CH₂ FH₄ or (6R)-FH₄ enantiomer, or byTS-FdUMP-CH₂ FH₄ due to very rapid ternary complex formation (Lockshinet al., Biochem. Pharmacol. 30:247-57 (1981)) prior to the 10-min biopsysample and one-carbon folate metabolism. In fact, the last explanationmay be the most attractive, since the maximum TS inhibition was at thisfirst biopsy time point. The degree of TS inhibition, 80.2% decreaseover baseline value, and relatively limited duration of TS inhibitionwould predict that higher concentrations of FdUMP (as would result from5-FU given shortly before, or with the CH₂ FH₄) would lead to thedesired therapeutic objective of complete TS inhibition.

In patient K. H., a fifty-five year old man with locally unresectableadvanced rectal adenocarcinoma, the TS pharmacodynamic tumor tissueanalyses were done twice, nine days apart. Following study, K. H.continued to receive intermittent bolus 5-FU. This patient had beenpreviously a partial responder to 5-FU plus LV, with stable disease atthe time of initial CH₂ FH₄ administration. There were modifications ofthe CH₂ FH₄ formulation between the 2 pharmacodynamic studies (See TableIII). In the first study week, the pH was not adjusted up from 2.0,after DEAE column isolation of the Sigma (6R,S)-CH₂ FH₄. Thus, some ofthis folate may also have been 5,10-methenyl-tetrahydrofolate. In thesecond study week, the pH was adjusted up to 9.0, and no DEAE step wasused (with therefore 6 mM formaldehyde being present in the 40-cc volumefor injection).

Patient K. H. showed changes in TS and in FBC assays after CH₂ FH₄administration that were qualitatively similar to those of Patient A.M.,shown in Table III. Again, significant inhibition of TS over baselinevalues occurred in tumor samples after the CH₂ FH₄ was given, in theabsence of recent 5-FU exposure. On the first occasion, however, the pHof the formulation was low, and possibly the CH₂ FH₄ was less wellsolubilized (or less stable, or both) than on Week #2, when an alkalinepH was used in addition to an excess of CH₂ O. Comparison with patientA.M. suggests that the acute TS decrease resulted from FH₄ rather thanCH₂ FH₄. As in Patient A.M., TS inhibition, on both occasions, wastransient, averaging 36 to 44% of baseline values for the combined dataof the two studies, during the 20 to 30 min period after CH₂ FH₄ wasgiven. The most significant evidence of an increase in CH₂ FH₄, asreflected by FBC assay, was at 24 hr after the first dose, which wasexpected on the basis of slow polyglutamation of folates generally.Significant drops in FBC also occurred in both weeks of study, againsuggestive of the postulated one-carbon exchange between FH₄-monoglutamates and endogenous CH₂ FH₄ -polyglutamates. The fact of aless striking change in FBC values in tumor biopsies from K.H. than inA.M. is also consistent with the lower baseline FBC values (given in rawDPM, multiply by 0.0002 to convert to nmol/g units comparable to PatientA.M.), and the less striking but highly significant TS inhibition intumor of K.H. As with Patient A.M., the data would predict, using purelykinetic arguments, that higher FdUMP levels generated from 5-FU givencloser to the time of CH₂ FH₄ dosing would lead to desired abrogation ofTS activity.

It has long been known that FdUMP tends to persist at low levels intissues following a single dose of 5-FU. FdUMP may therefore be slowlyreleased from the RNA storage compartment inside cells.

Thus, because only trace concentrations of FdUMP are required to inhibitTS, if CH₂ FH₄ or FH₄ levels are high, the TS inhibition observed inthese two patients was likely to have been due to facilitation by thenatural (6R)-CH₂ FH₄ or (6S)-FH₄ enantiomers (diastereomers) of the CH₂FH₄ formulation on TS binding by residual FdUMP levels. These resultssuggest that repeated administration of CH₂ FH₄ or FH₄ may be aseffective as repeated dosing with 5-FU, but without the toxicity ofdose-escalation of 5-FU.

The patients who received CH₂ FH₄ showed no acute toxicities due to thistreatment, including the instance of week #2 in K.H. when a slightexcess of CH₂ O was present in the preparation. However, they didcontinue to manifest the same toxicities as their prior experience with5-FU plus LV (i.e., mild nausea and fatigue). Patient A.M., as notedabove, had extremely advanced gastric cancer at the time of the studyand so was not evaluable for response. However, patient K.H. showedendoscopic evidence of continued disease stabilization if not at leastadditional, minor tumor regression noted over the subsequent monthsafter the two weeks of CH₂ FH₄ administration.

EXAMPLE 4 (6R,S)-FH₄ Administration to Rats Bearing Transplanted HepaticColonic Carcinomas

Table IV (below) shows the results of (6R,S)-FH₄ (see FIG. 3)administration to rats bearing transplanted hepatic colonic carcinoma.The present inventors have considerable experience with this model, andthe antitumor effects of 5-FU shown are typical results, as are the TSand folate assays of control and 5-FU-only-treated rats. A strikingfinding was of growth stimulation yet decreased TS levels after(6R,S)-FH₄ alone. In fact, the "free TS" levels in the (6R,S)-FH₄-only-treated rats were the lowest of all arms of the study. Thisobservation suggests that either the natural 6S-FH₄ or the unnatural6R-FH₄ may have formed TS-inhibitory TS-dUMP-folate ternary complexes.

Alternatively, and more likely (since TS was assayed 24 h after drugexposure), (6R,S)-FH₄ caused decreases in TS levels on the basis ofchanges in gene regulation. Thus, the growth stimulation by FH₄ may havebeen by a purine pathway effect (vs. by TS which is the rate-limitingenzyme for pyrimidine pathway de novo DNA synthesis). These biochemicalresults are consistent with the inventors' hypothesis that (6R,S)-FH₄potentiates 5-FU antitumor effect by increasing the degree of"unbalanced growth" resulting from TS inhibition in the face of on-goingpurine synthesis. In combination, the degree of synergy of (6R,S)-FH₄with 5-FU in this example is greater than previously found for(6R,S)-leucovorin (Carlsson et al., Anticancer Res. 10:813-16 (1990)).

                  TABLE IV                                                        ______________________________________                                        (6R,S)-TETRAHYDROFOLATE.sup.a AS A MODULATOR OF                               5-FU IN AN EXPERIMENTAL LIVER CANCER IN RATS.sup.b                            RESULTS AT DAY 17 AFTER TRANSPLANTATION                                       (Average of 3 rats/treatment)                                                          TUMOR     TS.sup.d                                                   TREAT-   WEIGHT    (p       5,10-CH.sub.2 FH.sub.4 .sup.d                                                           FH.sub.4 .sup.d                         MENT     (g)       mole/g)  (nmol/g)  (nmol/g)                                ______________________________________                                        CONTROL  5.84      18.96    0.69      1.18                                    5-FU ONLY                                                                              1.03      9.03     4.11      2.39                                    (30 MG/KG)                                                                    5-FU.sup.c +                                                                           0.31      9.23     1.23      1.76                                    (6R,S)-FH.sub.4 .sup.c                                                        (6R,S)-FH.sub.4                                                                        10.43     7.13     2.93      2.31                                    only                                                                          (30 mg/kg)                                                                    ______________________________________                                         .sup.a (6R,S)-FH.sub.4 was the commercially available racemic                 tetrahydrofolate from Fluka Chemical Corp. (Cat. No. 87355,                   "Tetrahydrofolic acid dihydrochloride monohydrate," or                        "5,6,7,8Tetrahydropteroyl-L-glutamic acid dihydrochloride monohydrate,"       >94% by HPLC). The (6 R,S)FH.sub. 4 was weighed, dissolved in normal          saline, and injected daily by tail vein administration using the airfree      Protector device to prevent oxidative destruction of the folate.              .sup.b Inoculation of 1 × 10.sup.6 viable colon tumor                   (nitrosoguanidineinduced) cells under the liver capsule on Day 1 (Carlsso     et al., Anticancer Res. 10:813-16 (1990)). Animals sacrificed on Day 17       for excision of single liver tumor nodules for pharmacodynamic studies.       .sup.c 30 mg/kg                                                               .sup.d Assays done as described (Spears et al. Adv. Exp. Med. Biol.           244:98-104 (1988)) and done at 24 h after last injection.                

The above antitumor results have been confirmed in a separateexperiment, using the same tumor line, dose, and route of administrationof 5-FU and (6R,S)-FH₄ to 6 rats/group. Animals receiving (6R,S)-FH₄alone showed tumor growth enhancement (171% of control tumor weight), to6.0±1.1 g, while the 5-FU+(6R, S)-FH₄ combination resulted in 0.70±0.10g size tumors.

EXAMPLE 5 Spontaneous Conversion of CH₂ FH₄ to FH₄ by Dilution

FIG. 4 shows the results of TS-[³ H]FdUMP-folate binding assay of CH₂FH₄ as a function of concentration of the folate in 0.2M Tris buffer, pH7.4, with and with formaldehyde (CH₂ O), 6 mM, addition. The CH₂ FH₄ wasprepared as the racemic (6R, S) material from (6R, S)-FH₄ and excessformaldehyde, and DEAE-column isolation as described in FIG. 1. Thispreparation was essentially free of free formaldehyde based oncolorimetric assay of bulk material (Nash, Biochem. J. 55:416-21(1953)).

At all concentrations (total assays volume 150 μl ), excess formaldeydewas required to obtain maximal binding (which was still only 19.3% ofstoichiometric binding). A notable effect was the increasing need forformaldehyde addition with increasing dilution, to obtain maximal CH₂FH₄ assay recovery.

This phenomenon has been a repeated observation in the laboratories ofthe inventors, and clearly shows that CH₂ FH₄ on dilution becomes FH₄with liberation of free formaldehyde (and formaldehyde solutionintermediates such as CH₂ (OH)₂). The concentration requirement forformaldehyde to reverse the FH₄ formation caused by dilution is in themillimolar range which is vastly higher than physiologic concentrationsof formaldehyde (which possibly does not occur in any free state).

This requirement for a large excess of formaldehyde to shift theequilibrium between FH₄ and CH₂ FH₄ (Eq. 1) was found by the inventorsto

    CH.sub.2 FH.sub.4 =FH.sub.4 +CH.sub.2 O                    Eq. 1

be independent of temperature, pH or the formaldehyde content ofcharcoal isolation, the presence of air exposure, or the presence ofreducing agents. In addition, [11-¹⁴ C]CH₂ FH₄ prepared as described(Moran et al., Proc. Natl. Acad. Sci. USA 76:1456-60 (1979)), andDEAE-purified (as the concentrated material) of excess ¹⁴ CH₂ O, wasconfirmed to have a labile ¹⁴ CH₂ O group by dimedone trapping. Forinstance, 46,664 DPM of [11-¹⁴ C]-CH₂ FH₄ diluted to 1 ml in H₂ O wasfound to have 67.8% of the label recoverable by chloroform extraction ofdimedone (methone) product (37° C.).

EXAMPLE 6 Pharmacologically Mediated Decreases in Intratumoral FH₄ inPatients Receiving 5-FU Plus High Dose (6R,S)-Leucovorin are Associatedwith Rapid Reversal of Antitumor Effects

As noted previously (see pages 5-6) and mentioned in C. P. Spears, etal., Proc. Am. Soc. Clin. Oncol. 8:69 (1989), the inventors have usedpharmacologic intervention to convert intracellular FH₄ to CH₂ FH₄ inpatients receiving high-dose reduced folate therapy combined with weeklyi.v. bolus 5-FU, 450 mg/sq. m. The method used was administration ofi.v. L-serine, 15.0 grams, over 1 hour after the end of overnighthigh-dose (6R,S)-leucovorin i.v. infusion.

In contrast to the data shown in Tables II and III, the inventors wereable to also assay for intratumoral FH₄ in addition to CH₂ FH₄ (thislatter is also referred to as "FBC" in those Tables or Folate-BindingCapacity), at time points during this pharmacologic forcing ofmetabolism of FH₄ to CH₂ FH₄ through the enzyme, serinehydroxymethyltransferase. This enzyme splits a carbon of L-serine togenerate formaldehyde in the presence of FH₄.

Table V below details the results of pharmacodynamic studies in 2patients (at Norris-USC Comprehensive Cancer Center, Los Angeles).

                  TABLE V                                                         ______________________________________                                        Effects of Conversion of FH.sub.4 to                                          CH.sub.2 FH.sub.4 in Breast Cancer Biopsies                                   TIME.sup.a                        CLINICAL                                    (min)     CH.sub.2 FH.sub.4                                                                      FH.sub.4 .sup.b                                                                       RATIO.sup.c                                                                          EFFECT                                      ______________________________________                                        Patient                                                                             0       0.194    2.370 12.2   Accelerated                               H.S..sup.d                                                                          22      0.253    0.854 3.4    progression of                                  65      0.340    0.907 2.7    tumor growth                              Patient                                                                             0       0.265    1.943 7.3    Accelerated                               M.H..sup.e                                                                          25      0.396    1.128 2.9    progression of                                  82      0.475    1.420 3.0    tumor growth                              ______________________________________                                         .sup.a Minutes after initiation of 15.0 gram LSerine i.v. 60min infusion,     after end of overnight (6R,S)leucovorin highdose i.v. infusion.               .sup.b Assays performed as described (C.P. Spears et al., Adv. Exp. Med.      Biol. 244:97-106, 1988).                                                      .sup.c Ratio of FH.sub.4 /CH.sub.2 FH.sub.4.                                  .sup.d A 74year-old woman with advanced chestwall infiltrating breast         carcinoma metastases, with stable disease activity on weekly 5FU plus         highdose (6R,S)leucovorin.                                                    .sup.e A 43year old woman with advanced chestwall metastases who has          previously experienced a minimal response to weekly 5FU plus highdose         (6R,S)leucovorin.                                                        

Similarly, in a third patient (a 42-year old woman with extensive boneand soft tissue metastases in whom no biopsies were taken), L-serinei.v. infusion added to 5-FU/(6R,S)-leucovorin treatment resulted inrapid acceleration of objective tumor growth and tumor symptoms.Subsequently, in order to reverse the folate conversion, toward FH₄ byglycine administration, 10 grams i.v., this patient then had a rapidresponse to 5-FU therapy. The present inventors have previously shownthe effectiveness of glycine in promoting FH₄ production from CH₂ FH₄(Spears et al., Pteridines and Folic Acid Derivatives, (Curtius, H.-C.,et al., eds.) Walter de Gruyter, Berlin, pp. 811-816 (1990). Glycineacts as a trap for the methylene (formaldehyde unit) of CH₂ FH₄ in thepresence of serine hydroxymethyl-transferase, by converting theformaldehyde to the 3-carbon of L-serine.

This example supports the hypothesis that an objective of (6R,S)-CH₂ FH₄administration is expansion of pools of FH₄ (and its polyglutamates),and of increased ratios of FH₄ /CH₂ FH₄.

EXAMPLE 7 Cell Culture Study Comparing Relative Effectiveness ofDifferent Folate Forms on Assayed Intracellular Levels Of CH₂ FH₄ andFH₄

In further support of the hypothesis that objectives of CH₂ FH₄administration are increases in FH₄ and FH₄ /CH₂ FH₄ values, are theresults of a cell culture study, shown in Table VI, comparing therelative effectiveness of different folate forms on assayedintracellular levels of the two folates. Each folate form was dosed at10 μM in the natural diastereomer (enantiomer or diastereomer). The(6R)-CH₂ FH₄ (natural diastereomer) was free of excess formaldehyde, andstudied for only a 90-min effect vs. 16-hr for the other folates becauseof its lower extracellular stability.

                                      TABLE VI                                    __________________________________________________________________________    CH.sub.2 FH.sub.4 and FH.sub.4 Levels In CCRF-CEM Cells After 16-hr           Exposure to 10 μM Folate: Comparison of Sources                            (Intracellular Folates In nmole/10.sup.9 Cells, AVE ± S.D.                 CONTROL     (6R,S)-MTHF.sup.a                                                                     (6S)-LV.sup.b                                                                      DHF.sup.c                                                                         (6R)-CH.sub.2 --FH.sub.4 .sup.d                  __________________________________________________________________________    CH.sub.2 FH.sub.4                                                                  0.04   0.51    0.13 <0.02                                                                             0.19.sup.a                                            ±0.01                                                                             ±0.07                                                                              ±0.02                                                                           ±0.01                                                                          ±0.02                                         FH.sub.4                                                                           3.08   10.55   10.64                                                                              7.10                                                                              8.81.sup.a                                            ±0.13                                                                             ±0.59                                                                              ±0.25                                                                           ±0.01                                                                          ±0.39                                         __________________________________________________________________________     .sup.a Racemic methyltetrahydrofolate, 10 μM in each diastereomer.         .sup.b Natural (6S)leucovorin.                                                .sup.c Dihydrofolate.                                                         .sup.d The natural diastereomer, prepared as described in Example 1.          Results represent values at 90 min of exposure.                               Two observations are most notable in Table VI: (a) the brief (90 min)         (6R)CH.sub.2 FH.sub.4 exposure was as effective as 16hr exposure to the       other folates, and (b) the major effect of (6R)CH.sub.2 FH.sub.4 exposure     was to increase FH.sub.4 and FH.sub.4 /CH.sub.2 FH.sub.4 levels, with onl     a minor effect on CH.sub.2 FH.sub.4 levels per se.                       

EXAMPLE 8 Comparison of Natural (6R)-CH₂ FH₄ and Unnatural (6S)-CH₂ FH₄Diastereomers

A study has been completed, for comparison of natural (6R)-CH₂ FH₄ andunnatural (6S)-CH₂ FH₄ diastereomers. The preparation of the two formswas done as described by Kisliuk, et al., Cancer Treat. Rep. 61:647-50(1977), using DEAE-cellulose column chromatography, with elution bypotassium bicarbonate buffer. The two separate major peaks wereindividually pooled, lyophilized to dryness, and used for injection. Asin the original detailed description of the method of Kisliuk, et al.(Biochem. 20:929-934), the methylene content was substoichiometric withtetrahydrofolate (FH₄). That is, methylene units were present inapparently lower concentration than FH₄ concentrations.

Rats bearing the transplanted colon cancer, as described in Example 4and in Carlsson, et al., Anticancer. Res. 10:813-816, 1991, were treatedwith either the (6R)-CH₂ FH₄ or the (6S)-CH₂ FH₄, formulated andinjected according to the methods described in Table IV, except that theinjected amounts were 15 mg/kg per dose.

At Day 17, the rats were sacrificed, and the tumors were measured forboth volume end weight. Volumes were calculated by the equation for anellipsoid in revolution, V=(4/3)πab² were a=the long diameter/2 andb=the perpendicular short diameter/2.

FIG. 5 shows the individual tumor results of this experiment. The filledcircles are the weights and volumes of tumors of rats treated with theunnatural (6S)-CH₂ FH₄ diastereomer (mostly (6R)-FH₄ material in dilutesolution and blood); the triangles are results for the natural (6R)-CH₂FH₄ (mostly (6S)-FH₄); and the open circles, control non-treated tumors.It is emphasized here that no fluorinated pyrimidine was used, just thefolate injections. The average values (±S. E.) of these data werepresented in Table VII.

                  Table VII                                                       ______________________________________                                                   Weight ±        Volume ±                                     Folate     SE        P-Value* SE      P-Value*                                ______________________________________                                        Control    7.93      --       7.81    --                                                 ±2.42           ±0.15                                        (6S)-CH.sub.2 FH.sub.4 .sup.a                                                            2.14      <0.001   0.74    <0.03                                   (Unnatural)                                                                              ±0.27           ±0.29                                        (15 mg/kg)                                                                    (6R)-CH.sub.2 FH.sub.4 .sup.b                                                            9.11      0.6      12.58   <0.046                                  (Natural)  ±1.56           ±1.49                                        (15 mg/kg)                                                                    ______________________________________                                         *Comparision with control rats, based on differences between the means.       .sup.a No cofactor activity by L. casei/dUMP spectrophotometric assay;        TS[.sup.3 H]FdUMP-folate ternary complex formation without formaldehyde       addition was 38.5% of the value for the natural (6R)CH.sub.2 FH.sub.4 wit     6 mM formaldehyde added. (About 1 pmole of parent CH.sub.2 FH.sub.4 was       needed in the binding assay.) Furthermore, this unnatural folate showed       decreases in TS[.sup.3 H]FdUMP complex background on addition of              formaldehyde (6 mM), which is a striking kinetic difference from the          natural form of CH.sub.2 FH.sub.4 .                                           .sup.b Only 11.8 ± 7.9% of maximal L. casei TS[.sup.3 H]FdUMP-folate       ternary complex formation (using 6 mM formaldehyde concentration) which i     consistent with ternary complex formation by (6S)FH.sub.4 alone.         

In view of the results of Table VII, it is clear that the racemicmixture of (6R,S)-CH₂ FH₄, which is predominantly (6R, S)-FH₄ in dilutesolution, has antitumor activity by two different mechanisms: theunnatural (6R)-FH₄ (in solution; (6S)-CH₂ FH₄ as the parent powder withsubstoichiometric methylene) diastereomer has directtumor-growth-inhibitory activity; the natural (6S)-FH₄ (in solution,(6R)-CH₂ FH₄ as the parent powder) potentiates fluorinated pyrimidineaction by TS-FdUMP-FH₄ ternary complex formation (TS-inhibition) in theface of purine and RNA growth stimulation with promotion of unbalancedgrowth.

EXAMPLE 9 Effect of the Unnatural Diastereomer (6S)-CH₂ FH₄ on HumanLymphoblast Cells

The unnatural diastereomer, (6S)-CH₂ FH₄ prepared by DEAE-column,potassium bicarbonate separation from the natural diasteromer asdescribed in Example 8, was dissolved in 0.2M Tris-HCl buffer, pH 7.0,containing sodium ascorbate (100 mM) and 2-mercaptoethanol. Thispreparation was from the same lot of material described in Example 8. Asnoted in the footnote of Table VII, in the absence of supplementalformaldehyde, the yield of dextrancharcoal isolated complex was 38.5% ofthe value for the natural (6R)-CH₂ FH₄ (with added 6 mM formaldehyde).The presence of formaldehyde actually decreased the yield ofTS-[3H]FdUMP-ternary complex of the unnatural diastereomer to 23.3%.This behavior of the unnatural diastereomer is exactly opposite to thatof natural (6R)-CH₂ FH₄ which becomes (6S)-FH₄ on dilution and shows astriking increase in TS-[³ H]FdUMP-folate yield on formaldehyde added ingreat excess.

Separate cultures tubes at 37° C. with 1 ml each of CCRF-CEM humanlymphoblast cells, 4.6×10⁵ cells/ml, in exponential log phase growthwere exposed to the unnatural diastereomer, (6R)-FH₄ (made up from(6S)-CH₂ FH₄, 1.56 mM stock solution, with 32 microliters added to eachml of cells), 48 μM final concentration. Cells were incubated for 30,60, and 120 minutes of drug exposure, prior to addition of [5-³H]deoxyuridine (150,000 CPM of approx. 18-20 Ci/mmo. spec. act.) and 10min. 37° C. incubation prior to 3% charcoal addition for isolation oftritiated water. The amount of tritiated water is proportional to theactivity of TS in the intact cells (N. Kundu et al., J. Med. Chem.33:1975-79 (1990)).

Control cells, without (6R)-FH₄ /(6S)-CH₂ FH₄ exposure showed minimalincreases over the 2 hour time period consistent with cell growth: 2566,2662, and 2902 CPM at 30, 60 and 120 min.

The CCRF-CEM cells exposed to 48 μM (6R)-FH₄ /(6S)-CH₂ FH₄ showedstriking TS-inhibition; values of tritiated water production were 2004,1750, and 1146 CPM at 30, 60 and 120 min. of drug exposure. These valuesare 78.1%, 65.7% and 39.5% of those of the control, untreated cells.Thus, inhibition of TS activity in intact cells was 60.5% by the twohour time point of drug exposure.

The inventors believe that these results indicate that the unnaturalfolate caused TS inhibition. The time course of progressive TSinhibition was consistent with increasing intracellular polyglutamationof the (6R)-FH₄ /(6S)-CH₂ FH₄ folate with increasing affinity forbinding to and inactivating TS inside the cells. This possibility wassuggested in Example 3 as an explanation for TS inhibition in apatient's tumor after (6R,S)-CH₂ FH₄ (predominantly (6R,S)-FH₄ insolution) administration. Polyglutamates of the unnatural (6R)-FH₄ havebeen reported in a purely cell-free kinetic study (Kisliuk et al., J.Biol. Chem. 249:4100-03 (1974)) to be stronger TS inhibitors than themonoglutamate, and any oxidation of these folates to their dihydrofolatepolyglutamates would also be expected to inhibit TS (Lockshin et al., J.Biol. Chem. 254:12285-88 (1979)).

We claim:
 1. A composition comprising (6R,S)-FH₄ and 5-FU in amountseffective in combination to achieve substantially complete inhibition ofthymidylate synthase (TS), together with a pharmaceutically acceptablecarrier.
 2. The composition of claim 1 further comprising an agent thatstabilizes FH₄.
 3. The composition of claim 2 wherein the agent thatstabilizes FH₄ is an ascorbate salt.
 4. The composition of claim 2wherein the agent that stabilizes FH₄ is reduced glutathione.
 5. Thecomposition of claim 2 wherein the agent that stabilizes FH₄ isformaldehyde.
 6. In a method of inhibiting the growth of a tumor in apatient by administering 5-FU to said patient, the improvementcomprising administering (6R,S)-tetrahydrofolate (FH₄) in an amountsufficient in combination with the 5-FU as active agents to achievesubstantially complete inhibition of thymidylate synthase (TS) in apatient with a tumor sensitive to said combination of active agents. 7.The method of claim 6 wherein FH₄ is administered to said patient byprotracted, continuous intravenous infusion through a central venouscatheter.
 8. The method of claim 6 wherein FH₄ is administered to saidpatient concurrently with 5-FU.
 9. The method of claim 6 wherein FH₄ isadministered to said patient prior to the administration of 5-FU. 10.The method of claim 9 wherein FH₄ is administered to said patient 6-24hours prior to the administration of 5-FU.
 11. The method of claim 9wherein FH₄ is administered to said patient 1-3 hours prior to theadministration of 5-FU.
 12. The method of claim 6 wherein FH₄ isadministered to said patient subsequent to the administration of 5-FU.13. The method of claim 12 wherein FH₄ is administered to said patient1-10 days subsequent to the administration of 5-FU.
 14. The method ofclaim 12 wherein FH₄ is administered to said patient 1-6 hourssubsequent to the administration of 5-FU.
 15. The method of claim 6wherein FH₄ is administered to said patient intravenously,intraarterially or intraperitoneally.
 16. The method of claim 15 whereinFH₄ is administered in a dosage of 5-500 mg/m².
 17. The method of claim16 wherein FH₄ is administered in a dosage of 20-200 mg/m².
 18. Themethod of claim 16 wherein FH₄ is administered intravenously.
 19. Themethod of claim 18 wherein FH₄ is administered to said patient every 4-6hours.
 20. The method of claim 18 wherein FH₄ is administered to saidpatient once daily.
 21. The method of claim 18 wherein FH₄ isadministered to said patient once weekly.
 22. The method of claim 19wherein FH₄ is administered prior to the administration of 5-FU.
 23. Themethod of claim 19 wherein FH₄ is administered subsequent to theadministration of 5-FU.
 24. The method of claim 20 wherein FH₄ isadministered to said patient through a central venous catheter.
 25. Themethod of claim 6 wherein the concentration of FH₄ administered is from0.1 to 20 mg/ml in alkaline vehicles.
 26. The method of claim 6 whereinthe concentration of FH₄ administered is from 0.1 to 10 mg/ml inphysiologic saline.